Recombinant P. heparinus Chondroitinase B Protein, CF

• Reactivity: Ph
• Applications: Enzyme Activity
• Format: Carrier-Free

Recombinant P. heparinus Chondroitinase B Protein, CF Summary

• Details of Functionality: Measured by its ability to hydrolyze chondroitin sulfate B. The specific activity is >90,000 pmol/min/μg, as measured under the described conditions.
• Source: E. coli-derived p. heparinus Chondroitin B Lyase/Chondroitinase B protein
  Gln26-His506, with an N-terminal Met and 6-His tag
• Accession #: Q46079
• N-terminal Sequence: Inconclusive results, Met predicted. Protein identity confirmed by MS analysis of tryptic fragments.
• Protein/Peptide Type: Recombinant Enzymes
• Gene: Phep_0789
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Endotoxin Note: <1.0 EU per 1 μg of the protein by the LAL method.

Applications/Dilutions

• Dilutions:
  • Enzyme Activity
• Theoretical MW: 55 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
• SDS-PAGE: 52-56 kDa, reducing conditions

Packaging, Storage & Formulations

• Storage: Use a manual defrost freezer and avoid repeated freeze-thaw cycles.
  • 6 months from date of receipt, -20 to -70 °C as supplied.
  • 3 months, -20 to -70 °C under sterile conditions after opening.
• Buffer: Supplied as a 0.2 μm filtered solution in Tris and NaCl.
• Purity: >95%, by SDS-PAGE under reducing conditions and visualized by Colloidal Coomassie® Blue stain at 5 μg per lane
• Assay Procedure:
  • Assay Buffer: 50 mM Tris, 5 mM CaCl₂, pH 7.5
  • Recombinant P. heparinus Chondroitin B Lyase/Chondroitinase B (rP. heparinus Chondroitinase B) (6974‑GH)
  • Substrate: Chrondroitin Sulfate B (Sigma, Catalog # C3788), 25 mg/mL stock in deionized water
  • 96 well UV Plate (Costar, Catalog # 3635)
  • Plate Reader (Model: SpectraMax Plus by Molecular Devices) or equivalent
  1. Dilute rP. heparinus Chondroitinase B to 0.4 ng/μL in Assay Buffer.
  2. Dilute Substrate to 4 mg/mL in Assay Buffer.
  3. Load 50 μL of 0.4 ng/μL rP. heparinus Chondroitinase B into the plate, and start the reaction by adding 50 μL of 4 mg/mL Substrate. Include a Substrate Blank containing 50 μL of Assay Buffer and 50 μL of 4 mg/mL Substrate.
  4. Read at an absorbance of 232 nm in kinetic mode for 5 minutes.
  5. Calculate specific activity:

  Specific Activity (pmol/min/µg) = (Adjusted Vmax* (OD/min) x well volume (L) x 10^12 pmol/mol) / (ext. coeff** (M^-1cm^-1) x path corr.*** (cm) x amount of enzyme (µg))

  
  *Adjusted for Substrate Blank
  **Using the extinction coefficient 3800 M^-1cm^-1
  ***Using the path correction 0.32 cm
  Note: the output of many spectrophotometers is in mOD. Per Well:
  • rP. heparinus Chondroitinase B: 0.020 μg
  • Chondroitin Sulfate B: 200 μg

Notes

Coomassie is a registered trademark of Imperial Chemical Industries Ltd.


This product is produced by and ships from R&D Systems, Inc., a Bio-Techne brand.

Alternate Names for Recombinant P. heparinus Chondroitinase B Protein, CF

• Chondroitin B Lyase
• Chondroitinase B

Background

Chondroitinase B from P. heparinus is a depolymerizing lyase that is especially active on the sulfated polysaccharide dermatan sulfate (DS) (1, 2). DS is a sulfated polysaccharide that is abundantly located in the skin, but is also present in the blood vessels, heart valves, tendons, and lungs. DS may play roles in coagulation, cardiovascular disease, carcinogenesis, infection, wound repair, and fibrosis (3). DS with a repeating disaccharide unit of IdoA alpha 1-3GlcNAc is most closely related to chondroitin sulfate (CS) that has a repeating disaccharide unit of GlcA beta 1-3GlcNAc. Due to this similarity, DS was originally referred to as chondroitin sulfate B, and the DS-depolymerizing enzyme was named Chondroitinase B. DS contains more sulfate than CS at levels comparable to the anticoagulant drug heparin, which is the reason that purified commercial heparin is more likely to be contaminated by DS. Chondroitinase B can be used to specifically degrade and distinguish DS in glycosaminoglycan preparations (4, 5, 6).

1. Oike, Y. et al. (1980) Biochem. J. 191:193.
2. Prabhakar, V. et al. (2009) J. Biol. Chem. 284:974.
3. Trowbridge, J.M. and Gallo, R.L. (2002) Glycobiology 12:117R.
4. Gu, K. et al. (1995) Biochem. J. 312:356.
5. Tkalec, A.L. et al. (2000) Appl. Environ. Microbiol. 66:29.
6. Wu, Z.L. et al. (2011) Glycobiology 21:625.

Publications for Chondroitin B Lyase/Chondroitinase B (6974-GH)(5)

Publications using 6974-GH

• Ono, F;Takahashi, Y;Shimada, S;Mizumoto, S;Miyata, S;Nitahara-Kasahara, Y;Yamada, S;Okada, T;Kosho, T;Yoshizawa, T; Carbohydrate sulfotransferase 14 gene deletion induces dermatan sulfate deficiency and affects collagen structure and bowel contraction PloS one 2025-05-06 [PMID: 40327642] (Bioassay, Mouse)
• A Persson, E Vorontsov, G Larson, J Nilsson Glycosaminoglycan Domain Mapping of Cellular Chondroitin/Dermatan Sulfates Sci Rep, 2020-02-26;10(1):3506. 2020-02-26 [PMID: 32103093] (Enzyme Activity, enzymne activity, Human)
• A Persson, A Gomez Tole, E Vorontsov, W Nasir, D Willén, F Noborn, U Ellervik, K Mani, J Nilsson, G Larson LC-MS/MS characterization of xyloside-primed glycosaminoglycans with cytotoxic properties reveals structural diversity and novel glycan modifications J. Biol. Chem., 2018-05-08;0(0):. 2018-05-08 [PMID: 29739851] (Bioassay)
• Xyloside-primed chondroitin sulfate/dermatan sulfate from breast carcinoma cells with a defined disaccharide composition has cytotoxic effects in vitro J Biol Chem, 2016-05-12;0(0):. 2016-05-12 [PMID: 27226567] (Bioassay, Human)
• N Gouignard, M Maccarana, I Strate, K von Stedin, A Malmström, EM Pera Musculocontractural Ehlers-Danlos syndrome and neurocristopathies: dermatan sulfate is required for Xenopus neural crest cells to migrate and adhere to fibronectin Dis Model Mech, 2016-04-21;0(0):. 2016-04-21 [PMID: 27101845] (Enzyme Assay, Xenopus)

Bioinformatics

• Gene Symbol: Phep_0789
• Uniprot:
  • Q46079()